We propose to continue our studies on basement membrane (BM) structure, function, and pathology. We have developed monospecific antibodies against BM components, and have used them to localize, by electron microscopy, the distribution of these antigens in tissues. We have demonstrated that fibronectin and type V collagen are not intrinsic components of BM. We now propose to define the relationship of fibronectin of BM using monoclonal antibodies reacting with different functional domains of the fibronectin molecule. These monoclonal antibodies will be used in electron immunohistochemical studies. We have developed and characterized in rats, an embryo derived parietal yolk sac carcinoma (ED-PYS) that has rapid BM synthesis. Now we propose to use this system to study the synthesis of BM using radioactive tracers, immunoprecipitation and PAGE fluorography. The intracellular localization of 4 BM components will be determined in ED-PYS cells and in their normal counterpart, the embryonic PYS. The exocytotic pathway of BM components will be compared to that of pro-collagen type I in fibroblasts. The last part of these studies will examine if the known decrease in glomerular charge characteristic of the nephrotic syndrome is due to changes in the antigentic components of BM (type IV collagen, laminin, entactin and heparan sulfate). Rats will be made nephrotic by injection of the aminonucleoside of puromycin, and the BM components localized by electron immunohistochemistry in normal and nephrotic rats. To achieve precise quantitation, glomeruli will be isolated from normal and nephrotic rats and the concentration of these antigens in glomerular homogenates determined by ELISA. Our goal is to understand how the BM components are processed and assembled to form BM in normal conditions, so that we can start asking intelligent questions about BM changes in diseases such as: diabetic microangiopathy, nephrotic syndrome, and other glomerulopathies.
